Journal Article

Characterization of Mitochondrial Alternative NAD(P)H Dehydrogenases in Arabidopsis: Intraorganelle Location and Expression

Dina Elhafez, Monika W. Murcha, Rachel Clifton, Kathleen L. Soole, David A. Day and James Whelan

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 1, pages 43-54
Published in print January 2006 | ISSN: 0032-0781
Published online January 2006 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci221
Characterization of Mitochondrial Alternative NAD(P)H Dehydrogenases in Arabidopsis: Intraorganelle Location and Expression

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The intramitochondrial location of putative type II NAD(P)H dehydrogenases (NDs) in Arabidopsis was investigated by measuring the ability of isolated mitochondria to take up precursor proteins generated from cDNAs using an in vitro translation system. The mature proteins of NDA1, NDA2 and NDC1 were judged to be located on the inside of the inner membrane because they were protected from protease added after the mitochondrial outer membrane had been ruptured. In contrast, NDB1, NDB2 and NDB4 were not protected from protease digestion in mitochondria with ruptured outer membranes and were deemed to be located on the outside of the inner membrane. Expression of all ND genes was measured using quantitative reverse transcription–PCR (RT–PCR) to determine transcript abundance, and compared with expression of alternative oxidase, uncoupler proteins and selected components of the oxidative phosphorylation complexes. NDA1 and NDB2 were the most prominently expressed members in a variety of tissues, and were up-regulated in the early daytime in a diurnal manner. Analysis of array data suggested that NDA1 clustered closest to the gene encoding the P-subunit of glycine decarboxylase. Taken together with the diurnal regulation of NDA1 observed here and in other studies, this suggests that NDA1 plays a role in integrating metabolic activities of chloroplasts and mitochondria. NDA2, NDB2 and Aox1a were up-regulated in a coordinated manner under various treatments, potentially forming a complete respiratory chain capable of oxidizing matrix and cytosolic NAD(P)H. NDB1 and NDC1 were down-regulated under the same conditions and may be regarded as housekeeping genes.

Keywords: Alternative oxidase; Alternative NAD(P)H dehydrogenase(s); Mitochondria; Protein import; Respiration; Aox, alternative oxidase; CI51, 51 kDa subunit of complex I; GFP, green fluorescent protein; ND, alternative NAD(P)H dehydrogenase; PGDC, P-subunit of glycine decarboxylase; PK, proteinase K; RPL19, cytosolic ribosomal protein of the large subunit; RPS1 and RPS13, nuclear-encoded mitochondrial ribosomal proteins of the small subunit; RT–PCR, reverse transcription–PCR; TIM, translocase of the inner mitochondrial membrane; Ucp, uncoupling proteins

Journal Article.  7257 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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